Oxygenated analogs of botanic seed

ABSTRACT

An analog of botanic seed is disclosed which comprises a plant embryo preferably encapsulated, or at least in contact with, a hydrated oxygenated gel. The gel can be oxygenated by passing oxygen gas through a gel solution before curing the gel or by exposing the gel to oxygen gas after curing. The gel is preferably oxygenated by adding to an uncured gel solution a suitably stabilized emulsion of a perfluorocarbon compound or a silicone oil, which compounds are capable of absorbing large amounts of oxygen, and are non-toxic and inert. An analog of botanic seed can further comprise an outer shell at least partially surrounding the gel and embryo, thereby forming a capsule. The outer shell preferably is shaped to aid the radicle of a germinating embryo in protrusively rupturing the capsule, thereby facilitating successful germination and minimizing incidence of seedling malformation. Other shell materials are selected to provide requisite rigidity to the capsule while imparting minimal restriction to successful germination.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of copending U.S. Patent applicationSer. No. 07/604,656, now U.S. Pat. No. 5,236,469 filed Oct. 26, 1990.

FIELD OF THE INVENTION

This invention relates to a method for propagating plants. Moreparticularly, it relates to methods for producing plant reproductiveunits, each containing a propagated plant embryo, capable of being sownlike natural seeds.

BACKGROUND OF THE INVENTION

Modern agriculture, including silviculture, often requires the plantingof large numbers of substantially identical plants genetically tailoredto grow optimally in a particular locale or to possess certain otherdesirable traits. Production of new plants by sexual reproduction, whichyields botanic seeds, can be slow and is often subject to geneticrecombinational events resulting in variable traits in the progeny.Also, such crossing is time- and labor-intensive. Further, inbredstrains such as those used to perform such crosses often lack vigor,resulting in low seed productivity.

Despite the drawbacks of conventional crossbreeding by sexual means,botanic seeds produced by such methods have an important advantage inthat each seed comprises food-storage organs and protective structuresthat shelter the plant embryo inside the seed from the harsh soilenvironment and nurture the embryo during the critical stages of sowingand germination. Without such organs and structures, the plant embryowould be incapable of surviving in nature until it grew to seedlingsize.

In view of the disadvantages of producing large numbers of identicalprogeny plants by sexual means, propagation of commercially valuableplants via culturing of somatic or zygotic plant embryos has beenintensively studied. Such "asexual" propagation has been shown for somespecies to yield large numbers of genetically identical embryos eachhaving the capacity to develop into a normal plant. Unfortunately, theseembryos, which are produced under laboratory conditions, lack theprotective and nutritive structures found in seeds. As a result, theembryos must usually be further cultured under laboratory conditionsuntil they reach an autotrophic "seedling" state characterized by anability to produce their own food via photosynthesis, resistdesiccation, produce roots able to penetrate soil, and fend off soilmicroorganisms. Such extensive laboratory culture during severaldistinct stages in plant development is time-consuming,resource-intensive, and requires skilled labor.

Some researchers have experimented with the production of "artificial"seeds in which individual plant somatic or zygotic embryos areencapsulated in a hydrated gel. (As used herein, "hydrated" denotes thepresence of free water interspersed throughout the matrix of gelmolecules comprising the gel capsule.) This method evolved from otherwork showing that encapsulating seeds in hydrated gels can improvegermination in some species, especially since such gels can besupplemented with plant hormones and other compounds that aidgermination and improve seedling survival in the field. With respect toartificial seeds, reference is made to European Patent Application No.0,107,141 to Plant Genetics, Inc., published on May 2, 1984 (claimingpriority under U.S. Pat. No. 4,562,663, filed on Oct. 12, 1982),teaching that hydrated gels used to encapsulate plant embryos shouldpermit gas diffusion from the environment to the embryo and protect theembryo from abrasion. A suitable gel can be selected from alginates,guar gums, agar, agarose, gelatin, starch, polyacrylamide, and othergels. The gel can include additives such as plant nutrients, pesticides,and hormones. If necessary, the gel can be surface-hardened to conferfurther resistance to abrasion and penetration.

While a hydrated gel capsule seems to provide adequate moisture for aplant embryo and satisfactory protection against physical trauma in someinstances, it has a poor permeability to atmospheric gases, especiallyoxygen, necessary for survival and growth of the embryo. As a result,there has been some effort directed to increasing the amount of oxygeninside the capsule. U.S. Pat. No. 4,808,430 to Kuono disclosesencapsulating a seed in a hydrated gel along with an air bubble.Unfortunately, such a bubble actually contains a very small volume ofair which in many instances does not provide enough oxygen for propergermination. This is especially the case when such bubble-containingcapsules are stored for a length of time at room temperature. At roomtemperature, embryos of many types of plants respire, even if notactually germinating, which consumes oxygen. Since a hydrated gel is apoor absorber of atmosphere oxygen, the embryo in the seed soon becomesoxygen-starved despite a presumably initially adequate supply in thebubble. As a result, no oxygen is left after such storage to supportgermination.

The drawbacks of including an air bubble along with an encapsulated seedwould not be fully rectified by encapsulating an embryo or seed in afoamed gel containing multiple air bubbles. The actual area availablefor gas exchange between the surrounding atmosphere, the gel capsule,the air bubbles, and the embryo is still quite small in a foamed gel.Such a small area, in combination with the low transfer rate of oxygenbetween air and a hydrated gel, would yield too low a rate of oxygendelivery to the embryo, especially during germination when oxygenrequirements rapidly escalate.

Hence, there is a need for an analog of botanic seed comprising a plantembryo in contact with a hydrated gel having an elevated concentrationof oxygen.

SUMMARY OF THE INVENTION

In accordance with the present invention, an analog of botanic seed isprovided which comprises a plant embryo or other unit of totipotentplant tissue encapsulated, or at least in contact with, a hydratedoxygenated gel. The gel preferably also includes dissolved nutrients andother beneficial compounds such as vitamins, hormones, and sources ofcarbon and energy, which can be utilized by the germinating embryo forenhanced growth or improved probability of survival. Suitable gelsolutes are substantially non-phytotoxic and can be selected from anumber of different types such as, but not limited to, sodium alginate,agar, agarose, amylose, pectins, dextran, gelatin, starch, modifiedcelluloses, and polyacrylamide.

Embryos of different species of plants require different amounts ofoxygen to undergo germination. Hence, an "oxygenated" gel as used hereinhas a concentration of oxygen that is higher than the concentration ofoxygen, at standard temperature and pressure, that would otherwise beabsorbed from the atmosphere. An "oxygen-carrying" gel is a similar typeof gel containing any extraneously added oxygen-absorbing oroxygen-carrying substance. Therefore, an oxygen-carrying gel is a typeof oxygenated gel.

One way of achieving oxygenation is to bubble oxygen gas through a gelsolution before curing the gel. Alternatively, gel capsules can beoxygenated by exposure to oxygen, under pressure if necessary, aftercuring.

Oxygenation of the gel is preferably enhanced by adding to an uncuredgel solution a suitably stabilized emulsion of an oxygen-carrying oroxygen-absorbing compound, selected from the group consisting ofperfluorocarbons and silicone oils. Representative perfluorocarbonsinclude perfluorocycloalkanes, perfluoro(alkylcycloalkanes),perfluoro(alkylsaturated heterocyclics), and perfluoro(tert-amines).These types of compounds are capable of absorbing large amounts ofoxygen, and are also inert and substantially non-toxic.

The emulsion is preferably stabilized by adding a substantiallynon-phytotoxic surfactant to a mixture of the gel solution andperfluorocarbon or silicone. Representative surfactants include methyloxirane polymers, egg albumin, and other substantially non-phytotoxicsurfactants such as those for food or ingestible pharmaceutical use.

The concentration of perfluorocarbon (or silicone oil) can depend on theoxygen requirements of the plant species being encapsulated in the gel,the oxygen-carrying capability of the perfluorocarbon (or silicone oil)being used, the type of gel, or the size of the microdroplets comprisingthe emulsion. Generally, the concentration of the perfluorocarbon (orsilicone oil) in the gel is about 15% w/v or less.

The concentration of surfactant is dependent upon the surfactant beingused and the size of the microdroplets comprising the emulsion. As thediameter of the droplets in a unit volume of perfluorocarbon emulsion isdecreased, the surface area of the disperse phase is increased, andcorrespondingly more surfactant is required to suitably stabilize theemulsion. Generally, the concentration of surfactant is about 10% w/v orless.

Although the embryo need only contact the hydrated oxygenated gel, suchas by resting on a surface of such gel, the embryo is preferablyencapsulated in the gel. Encapsulation allows the resulting analog ofbotanic seed to be handled without the possibility of the embryo losingcontact with the gel.

An analog of botanic seed according to the present invention can alsoinclude a rigid outer shell for increased protection against desiccationand physical trauma. The outer shell can have a tapered or wedge-shapedend to facilitate emergence of the radicle during germination. The outershell preferably has an orifice or analogous feature, or readily breaksapart during germination, making it relatively easy for the embryonicradicle to burst from the analog during germination. The outer shell canbe fabricated from a variety of materials including, but not limited to,cellulosic materials, glass, plastic, cured polymeric resins, paraffin,and combinations thereof.

The outer shell can further comprise plural layers, where the innerlayer thereof can comprise a relatively compliant and water-impermeablecellulosic material and the outer layer can comprise a polymericmaterial having a high dry strength and a low wet strength.Alternatively, the inner layers can comprise a rigid shape such as anopen-ended cylinder, where at least a portion of said open ends iscovered with an outer-layer material having a high dry strength and alow wet strength.

Further alternatively, the outer shell can comprise a relativelycompliant cellulosic or analogous material, shaped to at least partiallyconform to the shape of the hydrated oxygenated gel capsule therein, andhaving at least one tapered end. The tapered end terminates with anorifice which is preferably covered with a polymeric material having ahigh dry strength and low wet strength.

Although the embryo-containing gel unit preferably contains nutrientsdissolved therein, it is possible to dissolve the nutrients in aseparate nutrient-containing unit in contact with the embryo-containinggel unit. The nutrient-containing unit can be comprised of anysubstantially non-phytotoxic substance that will allow nutrients thereinto be transferred via water to the embryo-containing unit.Representative substances include, but are not limited to, water, a gelsimilar to that in the embryo-containing unit, vermiculite, perlite, orany polymeric material that is non-toxic and will release the nutrientsreadily over a period of time. For example, the nutrients may bemicroencapsulated in a manner known in the art.

It is therefore an object of the present invention to provide analogs ofbotanic seed characterized by a high percent germination of plantembryos therefrom.

A further object is to provide such an analog comprising a unit oftotipotent plant tissue encapsulated, or at least in contact with, ahydrated oxygenated gel to provide sufficient oxygen to enable the unitof totipotent plant tissue to successfully germinate.

A further object is to provide such an analog containing an increasedconcentration of oxygen over the concentration of oxygen that wouldnormally be present in hydrated gels by absorption of oxygen from theatmosphere.

A further object is to provide such an analog with an outer shell forincreased protection of the gel and embryo from desiccation and physicaltrauma but which does not impede the supply of oxygen to the embryowhile allowing the embryo to burst through the outer layer duringgermination.

The foregoing objects and other features and advantages of the presentinvention will be more fully understood as the detailed descriptionthereof proceeds, particularly when considered together with theaccompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a cross-sectional view of one embodiment of an analog ofbotanic seed according to the present invention comprising an embryoencapsulated in a hydrated oxygenated gel.

FIG. 1B is a cross-sectional view of an alternative embodiment of theanalog of botanic seed shown in FIG. 1A.

FIG. 1C is a cross-sectional view of another alternative embodiment ofthe analog of botanic seed shown in FIG. 1A.

FIG. 2A is a cross-sectional view of an analog of botanic seed similarto that shown in FIG. 1A but also including an outer shell.

FIG. 2B is a cross-sectional view of an alternative embodiment of theanalog of botanic seed shown in FIG. 2A.

FIG. 2C is a cross-sectional view of another alternative embodiment ofthe analog of botanic seed shown in FIG. 2A.

FIG. 3A is a cross-sectional view of an analog of botanic seed usable ina mechanical sowing process.

FIG. 3B is a cross-sectional view of an alternative embodiment of theanalog of botanic seed shown in FIG. 3A.

FIG. 3C is a cross-sectional view of another alternative embodiment tothe analog of botanic seed shown in FIG. 3A.

FIG. 3D is an isometric view of the exterior of an alternativeembodiment to that shown in FIG. 3C.

FIG. 4 is a stepwise sequential diagram illustrating one form ofgermination pattern frequently observed with an analog of botanic seedaccording to the present invention, wherein the gel capsule remainsattached for a time to the hypocotyl of the germinating embryo.

FIG. 5 is a stepwise sequential diagram illustrating a second form ofgermination pattern frequently observed with an analog of botanic seedaccording to the present invention, wherein the gel capsule remainsattached for a time to the germinating embryo at the cotyledon region ina manner similar to that of a natural seed.

FIG. 6 is a cross-sectional diagram of the analog of the botanic seedevaluated as Treatment (2) of Example 4.

FIG. 7 is a cross-sectional view of an analog of botanic seed evaluatedas Treatment (4) of Example 4.

FIG. 8 is a cross-sectional view of an analog of botanic seed evaluatedas Treatment (6) of Example 4.

FIG. 9A is a bar graph showing the percent germination of radicles andhypocotyls from analogs of botanic seed, as evaluated after two weeks'incubation in Example 6.

FIG. 9B is a bar graph showing percent malformations observed ingerminating embryos after two weeks' incubation in Example 6.

FIG. 9C is a bar graph obtained after two weeks' incubation showinglengths of radicles and hypocotyls of germinating embryos as evaluatedin Example 6.

FIG. 9D is a bar graph similar to that of FIG. 9A except that the datawere obtained in Example 6 after five weeks' incubation.

FIG. 9E is a bar graph similar to that of FIG. 9B except that the datawere obtained in Example 6 after five weeks' incubation.

FIG. 9F is a bar graph similar to that of FIG. 9C except that the datawere obtained in Example 6 after five weeks' incubation.

FIG. 10A is a bar graph showing percent malformations of variousembryonic structures of several species of gymnosperms after germinationfrom capsules, as evaluated in Example 7.

FIG. 10B is a bar graph showing radicle and hypocotyl lengths of thegerminating embryos evaluated in Example 7.

FIG. 11A is a bar graph showing percent malformations observed inembryos, as evaluated in Example 8.

FIG. 11B is a bar graph showing lengths of radicles and hypocotyls ofthe germinating embryos evaluated in Example 8.

FIG. 12A is a bar graph showing percent malformations observed inembryos, as evaluated in Example 9.

FIG. 12B is a bar graph of lengths of radicles and hypocotyls observedin the germinating embryos of Example 9.

FIG. 13A is a bar graph of percent malformations observed in embryosgerminating from capsules as described in Example 10.

FIG. 13B is a bar graph of lengths of radicles and hypocotyls observedin the germinating embryos of Example 10.

DETAILED DESCRIPTION

The analog of botanic seed disclosed herein comprises a unit oftotipotent plant tissue having at least one surface in contact with acured, hydrated, oxygenated gel.

As used herein, "totipotent" refers to a capacity to grow and developinto a normal plant. Totipotent plant tissue has both the completegenetic information of a plant and the ready capacity to develop into acomplete plant if cultured under favorable conditions. Totipotent planttissue is obtainable from several areas of a plant, such as meristematictissue and plant embryonic tissue.

Meristematic tissue is comprised of undifferentiated plant cells thatdivide to yield other meristematic cells as well as differentiated cellsthat elongate and further specialize to form structural tissues andorgans of the plant. Meristematic tissue is located, for example, at theextreme tips of growing shoots or roots, in buds, and in the cambiumlayer of woody plants.

Plant embryonic tissue can be found (in the form of a "zygotic" embryo)inside a botanic seed produced by sexual reproduction. Also, plant"somatic" embryos can be produced by culturing totipotent plant cellssuch as meristematic tissue under laboratory conditions in which thecells comprising the tissue are separated from one another and urged todevelop into minute complete embryos. Alternatively, a process termed"cleavage polyembryony" known in the art can be induced during natural.embryo development in seed. For simplicity, totipotent plant tissue isreferred to herein simply as the "embryo", unless stated otherwise.

As used herein, a "unit" of plant meristematic tissue or plant embryonictissue is a piece of such tissue that can be individually handled,placed on or encapsulated in a gel according to the present invention,and which will develop into a germinant and ultimately a plant underfavorable conditions.

The material used to encapsulate the totipotent plant tissue is ahydrated gel. A "gel" is a substance that is prepared as a colloidalsolution and that will, or can be caused to, form a semisolid material.

As used herein, "hydrated" denotes water-containing. Such gels areprepared by first dissolving in water (where water serves as thesolvent, or "continuous phase") a hydrophilic polymeric substance(serving as the solute, or "disperse phase") that, upon curing"gelling"), combines with the continuous phase to form the semisolidmaterial. In other words, the water becomes homogeneously associatedwith the solute molecules without experiencing any substantialseparation of the continuous phase from the disperse phase. However,water molecules can be freely withdrawn from a cured hydrated gel, suchas by evaporation or imbibition by a germinating embryo. When cured,these gels have the familiar characteristic of compliant solids, like amass of gelatin, where the compliance becomes progressively less and thegel becomes more "solid" to the touch as the relative amount of water inthe gel is decreased.

In addition te being water-soluble, suitable gel solutes are neithercytotoxic nor substantially phytotoxic. As used herein, a "substantiallynon-phytotoxic" substance is a substance that does not interferesubstantially with normal plant development, such as by killing asubstantial number of plant cells, substantially altering cellulardifferentiation or maturation, causing mutations, disrupting asubstantial number of cell membranes or substantially disruptingcellular metabolism, or substantially disrupting other process.

Candidate gel solutes include, but are not limited to, the following:sodium alginate, agar, agarose, amylose, pectin, dextran, gelatin,starch, amylopectin, modified celluloses such as methylcellulose andhydroxyethylcellulose, and polyacrylamide. Other hydrophilic gel solutescan also be used, so long as they possess similar hydration and gelationproperties and lack of toxicity. Also, it is important to be able to addother substances such as plant nutrients or emulsified materials to agel without substantially interfering with gelling ability. Further, acured gel must have sufficient strength to maintain the integrity of thecapsule without the capsule being so durable that a germinating embryocannot penetrate it.

Gels are typically prepared by dissolving-a gel solute, usually in fineparticulate form, in water to form a gel solution. Depending upon theparticular gel solute, heating is usually necessary, sometimes toboiling, before the gel solute will dissolve. Subsequent cooling willcause many gel solutions to reversibly "cure" or become gelled. Examplesinclude gelatin, agar, and agarose. Such gel solutes are termed"reversible" because reheating cured gel will re-form the gel solution.Solutions of other gel solutes require a "complexing" agent which servesto chemically cure the gel by crosslinking gel solute molecules. Forexample, sodium alginate is cured by adding calcium nitrate (Ca(NO₃)₂)or salts of other divalent ions such as, but not limited to, calcium,barium, lead, copper, strontium, cadmium, zinc, nickel, cobalt,magnesium, and iron to the gel solution. Many of the gel solutesrequiring complexing agents become irreversibly cured, where reheatingwill not re-establish the gel solution.

The concentration of gel solute required to prepare a satisfactory gelfor encapsulation purposes according to the present invention variesdepending upon the particular gel solute. For example, a usefulconcentration of sodium alginate is within a range of about 0.5% w/v toabout 2.5% w/v, preferably about 0.9% w/v to 1.5% w/v. A usefulconcentration of agar is within a range of about 5% w/v to about 10%w/v, preferably about 8% w/v. (As used herein, the "% w/v" concentrationunit is equivalent to grams of solute per 100 ml of solvent.) Gelconcentrations up to about 24% w/v helve been successfully employed forother gels. In general, gels cured by complexing require less gel soluteto form a satisfactory gel than "reversible" gels.

It is preferable to provide the embryo with the usual spectrum of plantnutrients and other beneficial substances such as vitamins and a sourceof carbon and energy (herein collectively termed generally "nutrients")while the embryo is encapsulated in the gel. Typical ways of providingnutrients are to dissolve the gel solute in a solution of the nutrientsor to add a volume of concentrated nutrient solution to the gel solutionbefore curing the gel. In this way, when the gel cures, any areas of theembryo in contact with the gel are also in direct contact with nutrientsolutes, where the nutrient solutes are present in substantially uniformconcentrations throughout the gel. Another way to provide nutrients isto place a gel capsule containing the embryo but lacking nutrients incontact with a second mass of the same or a different type of hydratedgel which does contain nutrients. As a result of a nutrientconcentration gradient between the two hydrated gel masses, nutrientswill migrate from the nutrient-containing gel mass to theembryo-containing gel mass.

Another possible way to provide nutrients is to place a gel unitcontaining the embryo but lacking nutrients in contact with a secondunit comprising microencapsulated nutrients or nutrients associated withany substantially non-phytotoxic substance that will allow nutrientsdissolved therein to be transferred via water to the embryo-containinggel unit. Representative materials include, but are not limited to,water, a gel similar to the gel in the embryo-containing unit,vermiculite, perlite, or any polymeric material that is non-toxic andwill release the nutrients readily over a period of time.

A number of possible nutrient formulations exist in the art, including anumber of proprietary formulations. For example, a popular medium is the"MS liquid" (Murashige and Skoog, Physiologia Plantarum 15:473-497(1962)) containing the following dissolved in water:

    ______________________________________                                        NH.sub.4 NO.sub.3 1650    mg/L                                                KNO.sub.3         1900    mg/L                                                CaCl.sub.2.2H.sub.2 O                                                                           440     mg/L                                                MgSO.sub.4.7H.sub.2 O                                                                           370     mg/L                                                KH.sub.2 PO.sub.4 170     mg/L                                                Na.sub.2 EDTA     37.25   mg/L                                                FeSO.sub.4.7H.sub.2 O                                                                           27.85   mg/L                                                MnSO.sub.4.4H.sub.2 O                                                                           22.3    mg/L                                                ZnSO.sub.4.4H.sub.2 O                                                                           8.6     mg/L                                                H.sub.3 BO.sub.3  6.2     mg/L                                                KI                0.83    mg/L                                                Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                   0.25    mg/L                                                CuSO.sub.4.5H.sub.2 O                                                                           0.025   mg/L                                                CoCl.sub.2.6H.sub.2 O                                                                           0.025   mg/L                                                Glycine           0.2     mg/100 cm.sup.3                                     Nicotinic Acid    0.05    mg/100 cm.sup.3                                     Pyridoxine.HCl    0.05    mg/100 cm.sup.3                                     Thiamine.HCl      0.01    mg/100 cm.sup.3                                     Kinetin           0.1     mg/L                                                Myo-inositol      100     mg/L                                                IAA               10      mg/L                                                Sucrose           30000   mg/L                                                pH                5.7-5.8                                                     ______________________________________                                    

(Note: An "MS medium" will also contain 1.0% w/v agar. Murashige andSkoog, id.) Of course, when adding a nutrient solution to a gelsolution, the concentrations of both solutions should be high enoughsuch that the resulting mixture of the two solutions has the properconcentrations of gel and nutrients.

The nutrient solution can also include plant growth hormones and othercompounds serving to further increase the probability of germinantsurvival.

As used herein, a "nutrient liquid" is an aqueous solution of nutrientssimilar to the "MS liquid" formulation. A "nutrient agar" is similar tothe "MS medium." Changes in types and amounts of certain ingredients canbe made to meet the needs of specific types of plants without departingin any substantial manner from the purpose and utility of a nutrientliquid or nutrient medium.

Since nutrient media, nutrient liquids, and any nutrient-containing gelis a rich growth medium for microorganisms and fungi, it is importantthat all such liquids, as well as the embryos themselves, be sterilebefore use. Embryos are kept sterile by culturing under sterileconditions. Liquids can be autoclaved or microfiltered.

As used herein, an "oxygenated" gel has a concentration of oxygentherein that is higher than the concentration of oxygen at standardtemperature and pressure that would be present in the gel as a resultonly of absorption from the atmosphere. An "oxygen-carrying" gel as usedherein is one that has any extraneously-added oxygen-absorbing oroxygen-carrying substances.

Oxygenation of a gel can be achieved by several methods. First, a gelsolution can be oxygenated before curing by passing oxygen gas throughthe solution. On a laboratory scale, this is typically performed byplacing the solution in a "gas-washing bottle" known in the art andbubbling oxygen gas through the solution while the solution is in thebottle. Analogous methods can be employed for oxygenation of largevolumes and for oxygenation of a continuous stream of uncured gel. Whenoxygenating a gel solution in this manner, it should be kept in mindthat hot solutions generally absorb less oxygen than cold solutions.Second, a gel can be oxygenated after curing by, for example, placingthe gel in a pressurized oxygen or pure oxygen environment. Thesemethods are also effective when the gel contains oxygen-carrier oroxygen-absorbing compounds, discussed in further detail below.

The concentration of oxygen in an oxygenated gel will depend on a numberof factors. In terms of a lower threshold concentration, the oxygenconcentration in a gel capsule surrounding an embryo is preferably atleast adequate to support enough growth of the radicle (embryonicstructure that eventually becomes the plant root) for it to rupture thecapsule and become exposed to oxygen in the atmosphere. The radicle isvery sensitive to oxygen concentration. For example, if the oxygenconcentration in a gel capsule surrounding an embryo is too low, themeristematic tissue in the radicle dies before the radicle can grow outof the capsule (see Example 2). Generally, if the oxygen concentrationis high enough for growth of the radicle, it is also high enough tosupport protrusive growth of other parts of the plant embryo from thecapsule, such as the shoot. The lower threshold concentration of oxygenseems to depend in part on the particular plant species represented bythe embryo. Other determinants of the concentration of oxygen in a gelinclude the thickness of the gel, the fact that different types of gelsolutes will absorb different amounts of oxygen, the degree of hydrationof the gel, the concentration of the gel solute, presence or absence ofother solutes in the gel such as nutrients and concentrations thereof,and the temperature of the gel. Therefore, in most cases, the lowerthreshold oxygen concentration is best determined for a specific plantembryo and capsule configuration by performing a simple germinationexperiment involving a series of identically encapsulated embryos inwhich each gel capsule in the series has a stepwise different oxygenconcentration from all other capsules in the series.

In a preferred embodiment, the concentration and availability of oxygenin the gel are increased by including in the gel an oxygen-absorbing oroxygencarrying compound. Certain such compounds are so efficient atabsorbing oxygen from the atmosphere that oxygenating the gel usingoxygen gas is not necessary in some instances.

A preferred class of compounds for use in increasing the concentrationof oxygen in a gel are the perfluorocarbons (PFCs). These compounds areorganic compounds in which all hydrogen atoms have been replaced byfluorine atoms. They are nonpolar, colorless, odorless, non-toxic,heat-stable, and extremely chemically inert. Because gases such ascarbon dioxide and oxygen have a high solubility in PFCs, PFC compoundshave been studied for use as blood substitutes. A first representativegroup of suitable PFCs comprises the perfluorocycloalkanes andperfluoro(alkylcycloalkanes) such as perfluorodecalin. A secondrepresentative group comprises the perfluoro(alkylsaturatedheterocyclic) compounds such as perfluorobutyltetrahydrofuran. A thirdrepresentative group comprises the perfluoro(tert-amine) compounds suchas perfluorotributylamine.

Because PFCs are nonpolar, they are not miscible with aqueous liquidssuch as gel solutions. In order to combine a sufficient amount of a PFCwith an aqueous gel solution to be useful as an oxygen absorber orcarrier, it is necessary to create a suitably stable emulsion of thePFC. In such an emulsion, microdroplets of the PFC, comprising thedisperse phase, are uniformly suspended in the gel solution (thecontinuous phase). As used herein, a "suitably stable" emulsion is onein which the disperse phase remains suspended in the continuous phase atleast until the embryo has germinated from the capsule. To suitablystabilize the emulsion, a surfactant can be utilized. The emulsion canalso be suitably stabilized in some instances merely by curing the gel.

The emulsion microdroplets are created by various methods known in theart, including using a high-shear mixing apparatus or via ultrasonicmeans. In the case of high-shear mixers, generally the higher the shearforce imparted to the liquid mixture, the smaller the microdroplet size.In the case of ultrasonic devices, more ultrasonic energy must be pumpedinto the liquid mixture to achieve smaller microdroplet sizes.Representative ranges of microdroplet sizes are from about 100 μmdiameter to less than 1 μm. In general, the smaller the microdropletsize, the more efficient the oxygen absorption and transport through thegel, since suspensions of smaller microdroplets have a larger totalmicrodroplet surface area than suspensions of larger microdroplets.However, as a result of their greater surface area, suspensions ofsmaller microdroplets require more surfactant to render them suitablystable than emulsions of larger microdroplets.

Generally, the PFC concentration in a gel is about 25% w/v or less. Thepreferred concentration range of PFC in a gel is up to about 15% w/v.The optimal range will depend in part on the type of gel solute, theoxygen-carrying capability of the particular PFC, the size of theemulsion microdroplets, and the desired oxygen concentration in the gel.For example, the optimal concentration range of PFC in an emulsion withsodium alginate is within a range of about 7.5% w/v to about 12% w/v.Results of experiments investigating various levels of PFC and gelconcentration can be found in the Examples.

Although a number of different types of surfactants would be effectivein stabilizing an emulsion of PFC, the surfactant must be non-toxic tothe embryo. As a result, certain ionic surfactants, such as sodiumdodecyl sulfate, which easily disrupt cell membranes, are unsuitable(see Example 8). Other surfactants, such as egg albumin and non-ionicsurfactants such as the methyl oxirane polymers(poly(oxyethylene)poly(oxypropylene) block copolymers) work well. Anexample is Pluronic F-68 from BASF Corp., Parsippany, N.J. In general,any substantially non-phytotoxic surfactant or emulsifier usable forfood or ingestible pharmaceutical use would be satisfactory.

The maximum amount of surfactant required to achieve a suitablystabilized emulsion is generally about 10% w/v, but can be higher ifextremely small microdroplets of PFC are formed during emulsification.In other words, as the diameter of microdroplets in a unit volume of PFCemulsion is decreased, the surface area of the PFC disperse phase isincreased, and a correspondingly greater amount of surfactant isrequired to suitably stabilize the emulsion. The preferred range ofsurfactant concentration is from about 0.4% w/v to about 6% w/v. Thesurfactant is typically added to a suspension of uncured gel and PFCjust before creation of the emulsion.

An alternative oxygen-absorbing compound that can be incorporated as anemulsion into a hydrated gel is a silicone oil. Silicone oils areavailable in a number of viscosity values, where oils having a viscositywithin the range of about 0.65 to about 15 centipoise are preferred.These oils, like PFCs, are nonpolar, colorless, odorless, non-toxic,heat-stable, chemically inert, and have high oxygen solubility values.In fact, some silicone oils have higher oxygen solubilities than manyPFCs. Emulsifying a silicone oil in a gel solution is performed insubstantially the same way as emulsifying a PFC. As with PFCs, asurfactant is generally required to achieve a suitably stable emulsionof silicone oil. Also, the concentration of silicone oil in a gel isgenerally about 25% w/v or less.

After preparing the gel liquid, whether it includes emulsified PFC orsilicone oil or not, preparing units of cured gel for use in germinatingplant embryos can be done in a number of ways. The method chosen willdepend in part upon how the embryo will contact the gel. It is importantthat the embryo have contact with the gel, either directly or via anintervening water-permeable "bridge" such as filter paper. In general,the embryo can rest on a surface of an oxygenated gel, rest in apreformed hole in a block of gel, or be entirely encapsulated in thegel. In the first two methods, the gel is generally cured preformed intothe preferred shape, or can be formed as a larger cured mass and cut tosize before inserting the embryo. In the case of totally encapsulatingan embryo in the gel, it is preferable to insert the embryo in a unit ofgel having the desired volume before the gel is completely cured.

FIG. 1A is a cross-sectional view of an analog of a botanic seed 10 madeby totally encapsulating an embryo 12 in a hydrated oxygenated gelcapsule 14. One way to make such a capsule is to place the uncured gelmixture in a separatory funnel. The stopcock on the funnel is adjustedto form drops of the gel liquid in a slow stepwise manner. Whenever adrop forms at the tip of the separatory funnel, an embryo is insertedfully into the drop using sterile forceps. Then, the drop containing theembryo is either captured in a space conforming to the desired shape ofthe capsule for curing or, in the case of gels that must be complexed tocure, dropped into complexing solution until curing is complete.

FIG. 1B is a cross-sectional view of an analog of a botanic seed 20where a large portion 22 of the gel capsule is preformed. In FIG. 1B,the large portion 22 is shown in the shape of a cube, although othershapes will also suffice, such as spherical or ovoid. The larger portion22 has a bore 24, which can also be preformed or cut after forming, intowhich the embryo 12 is inserted. If desired, the bore 24 can be sealedwith a plug 26 after inserting the embryo 12. The plug 26 can be made ofan additional piece of cured gel or other suitable material such asparaffin or similar material.

As can be seen in FIG. 1C, it is also possible to make an analog ofbotanic seed 30 by preforming two opposing capsule halves 32a, 32bwhich, when pressed together to form a complete capsule 34, define acavity 36 for receiving the embryo 12. Again, although FIG. 1C shows acubic configuration, the general concept shown therein is adaptable to avariety of shapes.

It is readily ascertainable that variations on each of the threeembodiments shown in FIGS. 1A, 1B, and 1C can be made which are withinthe scope of an encapsulated embryo according to the present invention.

It is also readily apparent that the embodiments of FIGS. 1A, 1B, and 1Ccan be made via an automated process.

It is also possible to encase an analog of botanic seed comprising anembryo-containing gel capsule in a rigid shell to afford protection tothe gel capsule and to the embryo therein from physical injury,desiccation, and other adverse environmental forces. For example, FIG.2A shows a cross-sectional view of one embodiment of such an analog 40comprising an embryo 12, a capsule 42 comprised of a hydrated oxygenatedgel in surrounding relationship to the embryo 12, and an outer shell 44in surrounding relationship to the gel capsule 42. The outer shell 44can be made from a large variety of materials including, but not limitedto, a cellulosic material, paraffin, moldable plastic or cured polymericresin, or a combination of these and/or other materials characterized bynon-toxicity and suitable rigidity. However, the rigidity must not besuch that an embryo germinating from within would not be capable ofgrowing out of the analog 40 without fatal or debilitating injury.Hence, polymeric materials having a high dry strength and low wetstrength are particularly desirable. Also desirable are shell materialsthat break apart easily upon application of an outwardly protrusiveforce from inside the capsule but are relatively resistant tocompressive forces applied to the outside of the capsule. The outershell 44 preferably also has an opening 46 toward which the radicle 48of the embryo 12 is oriented so as to facilitate protrusive growth ofthe radicle 48 from the analog 40 during germination. Otherwise, theradicle could become trapped inside the analog 40 and be prevented fromsuccessfully germinating.

Another possible embodiment of a shell-encased embryo-containing gelcapsule is illustrated in FIG. 2B showing a cross-sectional view of ananalog of botanic seed 50. The analog 50 comprises an embryo 12 and acapsule 52 comprised of a hydrated oxygenated gel in surroundingrelationship to the embryo 12, where the capsule 52 is cast in an innershell 54 to create a particular shape, such as a cylinder. The innershell 54 can be cut, for example, from a plastic or cellulosic drinkingstraw or analogous material such as glass tubing. Then, thecapsule-containing inner shell 54 is coated or otherwise layered with anouter shell 56 similar to the outer shell 44 of FIG. 2A. Again, it ispreferable that the outer shell 56 include an opening 58 to easeprotrusion of the germinating radicle. It is also preferable that theouter shell 56 have a low wet strength and a high dry strength.

Yet another possible embodiment of a shell-encased embryo-containing gelcapsule is illustrated in FIG. 2C showing a cross-sectional view of ananalog of botanic seed 60. As in FIG. 2B, the analog 60 in FIG. 2Ccomprises an embryo 12, a capsule 52 comprised of a hydrated oxygenatedgel in surrounding relationship to the embryo 12, and a rigidcylindrical shell 62 similar to the inner shell 54 of FIG. 2B. Inaddition, a cap 64 of paraffin or other polymeric material is applied toat least the first end 66 to afford protection against desiccation andphysical trauma as well as to properly restrain the cotyledons tofacilitate normal germination. A second cap (not shown) similar to thefirst cap 64 can also be applied to the second end 68 for additionalprotection. If the shell 62 is made from a water-impermeable substance,it is preferable that the cap 64, especially if applied to both ends 66,68, be made from a water-permeable substance to ensure adequate waterpenetration to the embryo 12 to support germination.

In all the embodiments shown in FIGS. 1A-1C and FIGS. 2A-2C, thehydrated oxygenated gel preferably includes dissolved nutrients. Inaddition, for oxygenation, the gel preferably includes a suitablystabilized emulsion of an oxygen-absorbing or oxygen-carrier substancesuch as a PFC compound or silicone oil suspended therein. In mostinstances, a gel containing such an emulsion should be oxygenated bypassing oxygen gas through the gel before curing or afterward byexposure to oxygen gas after curing. Alternatively, at least for embryosof plant species requiring relatively low oxygen concentrations forgermination, the gel including such an emulsion would be able to absorbsufficient oxygen from the atmosphere to ensure a high rate of embryogermination without the need for an oxygen-charging step.

In addition, whenever an embryo-containing gel capsule is substantiallysurrounded by an outer shell, it is at least partially isolated from theatmosphere. As a result, the gel should contain an emulsion as describedabove and be oxygen-charged to ensure that a sufficient supply of oxygenis present in the gel to supply the needs of the embryo duringgermination.

The embodiments shown in FIGS. 1A-1C and FIGS. 2A-2C are merelyrepresentative examples of possible capsule geometries. Other geometriesand capsule configurations are possible. For example, FIGS. 3A-3C showcross-sectional views of three other embodiments wherein the capsulesare bullet-shaped. Although capsules having such a shape can be usefulfor mechanical sowing, that is not the principal intent of the bulletshape. Rather, a tapered "bullet" end of a capsule helps guide anembryonic radicle germinating from within the capsule to grow toward the"bullet" apex for ease of escape from the capsule. As with naturalseeds, the capsules can be sown in any orientation in a soil or the likewithout interfering with the normal geotropism of the radicle.

FIG. 3A shows schematically a "shelf" capsule 70 comprising a block 71of hydrated oxygenated gel which preferably contains a stable emulsionof PFC or silicone oil. The gel block 71 defines a shelf 72 on which isplaced an embryo 12 having a radicle 48 oriented toward the taperedfirst end 73 of the capsule 70. In addition, the capsule 70 is shownhaving an optionell separate nutrient unit 74 in contact with the gelblock 71 and containing plant nutrients. The nutrient unit 74 may haveany of a number of possible forms, including a hydrated gel containingdissolved nutrients, a mass of microencapsulated nutrients, a mass ofslowly-soluble nutrient compounds, and other possible embodiments.Alternatively (not shown), the gel block 71 could occupy a larger spacein the capsule 70 and also include nutrients dispersed throughout thegel block 71, thereby obviating the need for a separate nutrient unit74.

FIG. 3A also shows an outer shell 75 in surrounding relationship to theblock 71 and nutrient unit 74 as well as the embryo 12. To permit use ofcommonly available materials as the outer shell 75, such as tubularmaterials, the outer shell 75 preferably has a circular transversecross-section, giving the outer shell 75 a cylindrical shape with atapered first end 73 and a second end 76. The outer shell 75 can beconstructed of, for example, a cellulosic tubular material similar to apaper drinking straw. Other materials such as plastic are also suitable.The tapered first end 73 can be formed via radicle crimps 77 or otherconstriction method to reduce the diameter of the outer shell 75 at thetapered first end 73. The second end 76 can be similarly tapered (notshown) or it can be shaped as shown as a transverse circular flatcontiguous with the outer shell 75. The tapered first end 73 preferablyterminates with an orifice 78 toward which the radicle 48 is urged togrow by the tapered first end 73 during germination. If required, theorifice 78 can be occluded with a covering 79 comprised of a softmaterial such as paraffin or, preferably, any suitable material having ahigh dry strength and a low wet strength. Alternatively, the covering 79can be comprised of a material that breaks apart easily upon applicationof a protrusive force from inside the capsule.

During sowing (not shown), the capsule 70 can be deposited in soil oranalogous plant-growing medium in any orientation. In the instance wherethe covering 79 has a low wet strength, subsequent irrigation wouldmoisten and soften the covering 79 and allow the radicle 48 of thegerminating embryo 12 to escape from the capsule 70 into the soil.

FIGS. 3B and 3C schematically show alternative embodiments of thecapsule design shown in FIG. 3A. In FIG. 3B, an embryo 12 is fullyembedded in a block 81 comprising a hydrated oxygenated gel. The gelblock 81 preferably also comprises a suitably stabilized suspension ofPFC or silicone oil. A separate nutrient-containing unit 84 is showncontacting the gel block 81. However, as in the FIG. 3A embodiment, thenutrients can be included in the gel block 81, which obviates the needfor a separate nutrient unit 84. Surrounding the gel block 81 and thenutrient unit 84 is an outer shell 85 shaped similarly to the outershell 75 of FIG. 3A. The radicle 48 of the embryo 12 points toward thetapered first end 83 of the outer shell 85. The tapered first end 83terminates with an orifice 88 which is shown lacking the covering 79 ofFIG. 3A to further illustrate possible embodiment variations. The FIG.3B embodiment is preferred over the FIG. 3A embodiment because theembryo is secured against losing contact with the gel block 81 by beingfully encapsulated therein.

The FIG. 3C embodiment is similar to the FIG. 3B embodiment with respectto the bullet shape of the capsule 90, the nutrient unit 94, and theouter shell 95 having a tapered first end 93 which terminates with anorifice 98. However, the hydrated oxygenated gel block 91 in which theembryo 12 is embedded is shown as an ovoid shape rather than thecylindrical shape of the gel block 81 in FIG. 3B. The FIG. 3C embodimentillustrates that the embryo-containing gel block 91 can be formedseparately instead of being cast in the outer shell as suggested in FIG.3B. Again, for improved oxygenation, the gel block 91 preferablyincludes a suitably stable suspension of PFC or silicone oil. Also, theseparate nutrient unit 94 can be eliminated by incorporating thenutrients into the gel comprising gel block 91.

In the interest of clarity, FIGS. 3A and 3B show the tapered first ends73 and 83, respectively, located some distance away from the radicle 48.However, it is preferable, as shown in FIG. 3C, that the tapered firstend 93 be located as close as possible to the radicle 48. This ensuresthat, during germination, the radicle 48 has only a minimal distance toelongate inside the capsule 90 before being urged toward the orifice 98by the tapered first end 93. Otherwise, geotropism of an elongatingradicle may cause the radicle 48 to grow away from the tapered first end93 and make it difficult for the tapered first end 93 to urge theradicle to grow toward the orifice 98.

FIG. 3D shows the exterior of an alternative embodiment 90a of thecapsule 90 of FIG. 3C having an outer shell 95a, a tapered first end93a, and a second end 96a corresponding to similar features shown inFIG. 3C. In FIG. 3D, the tapered first end 93a has a flat crimp 99rather than the bullet-shaped configuration shown in FIG. 3C. As in FIG.3C, the embryo radicle (not shown) inside the capsule 90a of FIG. 3D isoriented toward the tapered first end 93a, particularly toward anopening 98a left in the crimp 99.

FIGS. 4 and 5 each show stepwise sequential images of a gymnospermembryo 12 germinating from an analog of botanic seed 100. Although theanalog 100 is shown comprising an ovoid-shaped hydrated oxygenated gelcapsule 101, FIGS. 4 and 5 are also applicable to other capsuleembodiments, such as those including an outer shell. For simplicity, theanalog 100 is shown being "sown" by placing on top of a soil surface102, even though in most cases the analog 100 would be sown beneath thesoil surface 102. Also, for clarity, each image except the rightmostimage in each of FIGS. 4 and 5 is shown as a cross-sectional view.

FIG. 4 shows a stepwise germination sequence of an embryo 12 from thecapsule 101 in which both the radicle 48 and the cotyledons 49 burstfrom different ends of the capsule 101 at substantially the same time.The first, or leftmost, image shows the capsule 101 containing an embryo12 embedded therein. In the second image, germination has begun and thegrowing radicle 104 has undergone sufficient growth to burst out of thecapsule 105. Also, the cotyledons 106 have undergone sufficient growthto just begin protruding from the capsule 105. In the third, or middle,image, a root 108 (which developed from the radicle) is shownpenetrating the surface 102 of the soil, and the cotyledons 110 havefurther elongated. The capsule 112 thus remains affixed to the hypocotyl114 in a manner similar to a bead on a string. In the fourth image, theseedling 116 has become more upright, the root 118 has grown longerdownward and the cotyledons 120 have begun to spread apart. The capsule122, however, remains attached to the hypocotyl 124. Finally, in therightmost image of FIG. 4, the capsule is shown having split into twohalves 126 and 128 and fallen off the seedling 130.

For purposes of comparison, FIG. 5 shows a germination pattern closelyresembling that of a natural seed. In the first, or leftmost, image, ananalog of botanic seed 100 is comprised of an embryo 12, having aradicle 48 and cotyledons 49, and a hydrogenated oxygenated gel capsule101 in surrounding relationship to the embryo 12. In the second image,the radicle 132 has burst from the capsule 134. In the third image, aroot 136 is shown penetrating the soil surface 102 and the cotyledons138 have elongated. The capsule 140 has a certain strength, such as asurface strength, sufficient to prevent the cotyledons 138 fromrupturing the capsule 140 during elongation while allowing the capsule140 to be pushed ahead of the growing cotyledons 138. In the fourthimage, the root 142 and cotyledons 144 have grown longer. The capsule146 remains attached to the cotyledons 144 while allowing them toelongate naturally without malforming. In the fifth image, the seedling148 has elongated sufficiently to elevate the capsule 150 off the soilsurface 102. Finally, in the rightmost image, the capsule 152 has fallenoff the cotyledons 154 in a manner similar to a seed husk of a naturalseed. The seedling 156 appears normal and has excellent prospects forfuture growth.

In the Examples below, a growth pattern such as that shown in FIG. 4wherein the capsule remains adhered to the hypocotyl of a germinatedembryo for a time is regarded as not as desirable as that shown in FIG.5 wherein the capsule remains attached for a time to the cotyledons in amanner similar to a natural seed. Nevertheless, there is no evidencethat a germination pattern as in FIG. 4 is in any way detrimental to thesurvival of the seedling. The germination patterns discussed above inrelation to FIGS. 4 and 5 have been regularly observed during numerousstudies of various embodiments of analogs of botanic seed according tothe present invention. While the pattern of FIG. 5 more closelyresembles that of a germinating natural seed, both the FIG. 4 and FIG. 5patterns will result in production of normal seedlings.

The following terms as used in the Examples are defined as follows:

"Somatic embryo" is a plant embryo that developed via the laboratoryculturing of totipotent plant cells or by induced cleavage polyembryony.

"Zygotic embryo" is a plant embryo removed from a seed of thecorresponding plant.

"Germinant" is an embryo that has undergone sufficient growth anddevelopment to protrude from a capsule, analogous to protruding from anatural botanic seed.

"Radicle" is that part of a plant embryo that develops into the primaryroot of the resulting plant.

"Cotyledon" refers generally to the first, first pair, or first whorl(depending on the plant type) of leaf-like structures on the plantembryo that function primarily to make food compounds in the seedavailable to the developing embryo but in some cases act as food storageor photosynthetic structures.

"Hypocotyl" is that portion of a plant embryo or seedling located belowthe cotyledons but above the radicle.

"Epicotyl" is that portion of the plant developed after germination fromthe stem apex.

"Capsule" refers at least to a hydrated gel in surrounding relationshipto a plant embryo embedded therein.

"Hypocotyl length" pertains to the length of the hypocotyl at the timethe hypocotyl was measured.

"Hypocotyl germination" denotes the emergence of the embryo shoot fromthe capsule, caused by elongation of the hypocotyl sufficiently to burstthe capsule. This term does not take into consideration any lengthcriteria or lack of hypocotyl malformations.

"Swollen hypocotyl" is an attribute of an abnormal embryo characterizedby the hypocotyl or a portion thereof having a greater than normaldiameter compared with hypocotyls on control bare embryos grown on thesurface of a nutrient agar or similar nutrient medium.

"Twisted hypocotyl" is an attribute of an abnormal embryo characterizedby the hypocotyl having grooves spiraling longitudinally up or down thelength of the hypocotyl. This defect is usually found only in embryosexhibiting swollen hypocotyls.

"Swollen cotyledons" is an attribute of an abnormal embryo of agymnosperm characterized by unusually large cotyledon(s) compared tocotyledons on control bare embryos grown on the surface of a nutrientagar or similar nutrient medium.

"Twisted cotyledon" is an attribute of an abnormal embryo of agymnosperm characterized by the cotyledon(s) having a spiraled ortwisted appearance.

"Radicle length" pertains to the length of the radicle at the time theradicle was measured.

"Radicle germination" denotes the emergence or protrusive growth of theroot from the capsule, caused by elongation of the radicle sufficient toburst the capsule. This term does not take into consideration any lengthcriteria.

"Growth through capsule" occurs when an embryo inside the capsuleundergoes elongation both of the radicle and the hypocotyl and burststhe capsule at both ends. This is usually evidenced by the capsuleremaining for a period of time as a captive spherical body around thehypocotyl.

"Normaicy" denotes the presence of all parts (radicle, hypocotyl,cotyledon(s), epicotyl) at time of evaluation, where, in the case ofgymnosperms, the radicle has length greater than 3 mm and no visiblydiscernable malformations compared to the appearance of control bareembryos grown on the surface of nutrient agar or similar nutrientmedium.

It is important to note that, as long as all parts of an embryo havegerminated, the corresponding germinant probably has the potential tobecome a normal seedling. We have no reason to believe thatmalformations evident in the following Examples below are fatal togerminants. Noting the quantity and quality of malformation is aconvenient way to comparatively evaluate the various methods and meansemployed for making analogs of botanic seed. Fortunately, plantembryonic tissue is exquisitely sensitive to non-natural conditions andmanifests that sensitivity in ways discernable to a trained observer.

EXAMPLE 1

This Example is an evaluation, for comparison purposes, of embryogermination from non-oxygenated capsules of the type as disclosed inEuropean Patent Application No. 0,107,141. (The European applicationreferred to herein as EPA '141 claims priority under U.S. U.S. Pat. No.4,562,663 filed on Oct. 12, 1982.) Individual sets of zygotic embryos ofNorway Spruce were subjected to one of the following Treatments:

Treatment (1): "Control" wherein bare embryos were placed directly onthe surface of nutrient agar in a manner known in the art.

Treatment (2): Embryos encapsulated in sodium alginate in the mannerdisclosed in EPA '141.

Treatment (3): Capsules lacking embryos were formed as disclosed in EPA'141, after which each capsule was cut in half, an embryo placed in thecenter thereof, and the capsule halves were pressed together around theembryo to seal the capsule around the embryo.

All Treatments were placed in covered Petri plates on the surface ofnutrient agar medium (1% agar). Six embryos were placed in each plateand six replicate plates were prepared for each Treatment. All plateswere placed in a 23° C. incubator under continuous filtered fluorescentlight to stimulate germination. After 28 days, the plates were removedfrom the incubator and examined for quality and quantity of germinants.

Upon examination, it was found that whole capsules according to EPA '141(Treatment (2)) did not allow the radicle to elongate. Although thehypocotyls of Treatment (2) embryos usually elongated, they weremalformed (twisted and swollen). These results indicate that the embryomust exert an excessive force injurious to the embryo in order togerminate from the EPA '141 capsule.

Embryos encapsulated by Treatment (3) split into halves under theprotrusive force of the germinating embryo, usually in the first twoweeks. However, the germinants still did not exhibit normal development.Nevertheless, a higher percent of the embryos receiving Treatment (3)germinated than observed with Treatment (2) embryos. Lack of normaldevelopment of Treatment (3) embryos was apparently not due to excessiverestraint imparted by the capsule since the capsules were seen to spliteasily.

Of the "Control" embryos of Treatment (1), 75% showed normalgermination. In contrast, of the Treatment (2) embryos, only 6% showednormal germination; and of the Treatment (3) embryos, only 14% showednormal germination. These results indicate that, although encapsulatingembryos in a more easily ruptured alginate capsule (Treatment (3))improved embryo germination, some other factor, such as lack of oxygenavailability through an unruptured capsule, seems to be responsible forthe poor embryo development seen with embryos encapsulated according toEPA '141, relative to bare embryos placed on nutrient agar having anunlimited exposure to oxygen.

EXAMPLE 2

This Example was an evaluation of whether the position of the embryowithin a gel capsule was a significant factor in determining the successrate of embryo germination and normal development.

Individual sets of zygotic embryos of Norway Spruce were subjected toone of the following treatments:

Treatment (1): "Control" wherein bare embryos were placed directly onthe surface of nutrient agar in a manner known in the art.

Treatment (2): Capsules lacking embryos were formed as an EPA '141,after which each capsule was cut in half, an embryo inserted thereinwith the radicle end positioned relatively close to the outer surface ofthe capsule compared with the shoot, then the capsule halves werepressed together around the embryo to reseal.

Treatment (3): As in Treatment (3) of Example 1.

All Treatments were placed in covered Petri plates on the surface ofnutrient agar medium. Six embryos were placed in each plate and sixreplicate plates were prepared for each Treatment. All plates wereplaced in a 23° C. incubator under continuous filtered fluorescent lightto stimulate germination. After 28 days, the plates were removed fromthe incubator and examined for quality and quantity of germinants.Results are tabulated in Table I.

                  TABLE I                                                         ______________________________________                                                 % Normal    Mean Length                                                                              Mean Length                                   Treatment                                                                              Germinants  Hypocotyls Radicles                                      ______________________________________                                        1 (Control)                                                                            81%         1.26 cm    1.44 cm                                       2 (Offset)                                                                             17%         0.63 cm    0.98 cm                                       3 (Centered)                                                                            8%         0.62 cm    0.72 cm                                       ______________________________________                                    

The data in Table I indicate the following conclusions:

(a) Embryos encapsulated with radicles situated close to the capsulesurface (Treatment (2)) yielded two times more normal germinants thanembryos encapsulated in the center of a capsule (Treatment (3)). Thisindicates that minimizing the protrusive force that must be exerted by agerminating radicle to burst from a capsule is beneficial to thegerminating embryo.

(b) Although mean hypocotyl lengths were about equal for Treatments (2)and (3), radicle length was longer for Treatment (2), indicating thatconditions for radicle growth were more favorable in Treatment (2).

(c) Poor radicle elongation in Treatments (2) and (3) appears to be dueto a limiting factor, such as low concentration of oxygen, prior tocapsule splitting. In instances where the radicle failed to elongate atall, a brownish mass of tissue formed on the radicle resembling acallus, indicating probable death of cells comprising the radicle tip.Although the capsules in Treatments (2) and (3) appeared to split easilyduring germination, they apparently did not split early enough toprevent tissue death. The fact that a larger percentage of radicles didelongate in Treatment (2) was probably due to a higher amount of oxygengetting to the radicle due to the split in the capsule.

EXAMPLE 3

This Example was an evaluation of the effects on embryo germination ofvarying the amount of surface area of zygotic embryos exposed to air.

Individual sets of zygotic embryos of Norway Spruce were subjected toone of the following treatments:

Treatment (1): "Control" wherein bare embryos were placed on the surfaceof nutrient agar in a manner known in the art.

Treatment (2): Bare embryos placed on the surface of a nutrient mediumcomprising complexed alginate (1.5% alginate) instead of agar.

Treatment (3): Embryos centrally encapsulated in blocks of nutrient agar(0.8% agar); blocks then placed on the surface of nutrient agar.

Treatment (4): Embryos encapsulated in blocks of nutrient agar (0.8%)with radicles protruding from the block; blocks then placed on thesurface of nutrient agar.

Treatment (5): Embryos encapsulated as in EPA '141 except that thealginate concentration was 1.5%, and a nutrient aqueous liquidcontaining dissolved nutrients as in "MS liquid" was used instead ofwater to dissolve the alginate; capsule diameter was about 3 mm;capsules then placed on the surface of nutrient agar.

To prepare alginate for Treatment (2), a 1.5% alginate solution wasprepared using a nutrient liquid similar to "MS liquid" and pouredslowly into sterile Petri dishes until the bottom of each dish wascovered. A solution of Ca(NO₃)₂ in the nutrient liquid was then sprayedinto the dishes using a plastic spray bottle to initiate complexing(gelling) of the alginate. After the alginate began to gel (about 3minutes), more Ca(NO₃)₂ solution in nutrient liquid was poured into eachdish to submerge the gelled alginate therein for about 20 minutes. TheCa(NO₃)₂ was then poured off and the complexed alginate rinsed withnutrient liquid for 5 minutes.

To prepare agar blocks for Treatments (3) and (4), blocks ofnutrient-containing agar were cut measuring about 4×4×5 mm using a smallspatula. Using sterile forceps, an embryo was inserted into each block,centered in the block for Treatment (3) and with the radicle protrudingoutside the block for Treatment (4). Embryos were inserted into theblocks radicle-end first for Treatment (3) and cotyledon-end first forTreatment (4). With Treatment (4), about half the embryo length was leftprotruding from the agar block.

Bare embryos (Treatment (1)) and encapsulated embryos (Treatments(2)-(5)) were placed on nutrient-agar surfaces in Petri dishes. Thedishes were covered and placed in a 23° C. incubator under continuousfiltered fluorescent light for 35 days. Subsequent examination revealedthe data shown in Table II.

                  TABLE II                                                        ______________________________________                                                               %                                                                             Germments % Germinants                                             % Normal   w/ Swollen                                                                              w/ Swollen                                   Treatment   Germinants Hypocotyls                                                                              Cotyledons                                   ______________________________________                                        1 (Agar control)                                                                          90%         0%       0%                                           2 (Alginate control)                                                                       8%        36%       0%                                           3            0%        91%       47%                                          4           61%        37%       26%                                          5           20%        75%       3%                                           ______________________________________                                    

The results and conclusions can be summarized as follows:

(a) The agar blocks with protruding radicles (Treatment (4)) produced61% normal germinants with radicle and hypocotyl lengths similar tocorresponding lengths of control embryos. This indicates that lack ofphysical restraint, free exposure to oxygen, and a nutrient supply areimportant for optimal growth of the radicle.

(b) The embryos encapsulated in alginate (Treatment (5)) produced only20% normal germinants. Fifty-nine percent of the radicles and 97% of thehypocotyls germinated but 74% of the hypocotyls were swollen andtherefore did not undergo normal development. This indicates that fullencapsulation in alginate presents at least one environmental restraintto normal germination, such as lack of oxygen.

(c) Bare embryos placed on the surface of complexed alginate (Treatment(2)) had the same amount of embryonic tissue exposed to air as thecontrol embryos placed on agar (Treatment (1)). Nevertheless, theTreatment (2) embryos experienced much less normal germination thancontrols. The reason for this is unclear.

(d) The embryos embedded completely inside nutrient agar blocks(Treatment (3)) therein yielded no normal germinants at all. Allhypocotyls germinated but 92% thereof were swollen. This indicates, asin Treatment (5), that complete encapsulation without providing oxygenappears to present an environmental impediment to successfulgermination.

EXAMPLE 4

This Example is an evaluation of germination performance observed withembryos of Norway Spruce individually inserted halfway into blocks ofnutrient agar medium versus embryos individually placed on the surfaceof a unit of nutrient gel medium, where each unit of the gel medium wasthen surrounded by a rigid protective "shell" made of either thintransparent plastic or glass.

Individual sets of zygotic embryos were subjected to one of thefollowing Treatments:

Treatment (1): "Control" wherein bare zygotic embryos were placed on thesurface of nutrient agar medium.

Treatment (2): As shown in FIG. 6, glass cylindrical capsule shells 160were made having length about 12 mm, outside diameter about 7 mm, andinside diameter about 5.6 mm. One end 161 of each shell was sealed withan elastomeric septum 162. After sterilization, the shells were orientedvertically openend up and filled about two-thirds full with nutrientagar medium 163. A zygotic embryo 164 was inserted halfway into theexposed agar surface 165 in each shell, cotyledon end 166 first, leavingthe radicle 167 exposed to the atmosphere. The resulting capsules 168were turned on their sides on a nutrient agar surface for incubation.

Treatment (3): Same as Treatment (2) except that, after inserting theembryos in the agar, the open ends of the glass shells were subsequentlypartially sealed from the atmosphere using PARAFILM (a registeredtrademark of American National Can, Greenwich, CT.) laboratory film (aparaffin film well-known in the art). The film was applied to the openend in a manner that left a small hole through which the radicle couldprotrude during germination.

Treatment (4): As shown in FIG. 7, rigid shells 170 were made by cuttinga 4 mm diameter clear plastic drinking straw to 4 mm lengths. Aftersterilization, each shell 170 was oriented horizontally and filled abouthalf full with nutrient agar medium 171, leaving a flat agar surface 172inside each shell extending the length of the shell. An embryo 173 wasplaced on the agar surface (or "shelf") inside each shell. One end 174of each shell was sealed using paraffin 175; the other end 176 was leftopen to the atmosphere, where the radicle 177 of the embryo 173 thereinpointed toward the open end 176. The resulting capsules 178 were placedon their sides on a nutrient agar surface for incubation.

Treatment (5): Same as Treatment (4) except that, after placing theembryo in the capsule, the open end of the shell was partially sealedusing PARAFILM in the same manner as described in Treatment (3).

Treatment (6): As shown in FIG. 8, rigid shells 180 were made by cuttinga 4 mm diameter clear plastic drinking straw to 8 mm lengths. Aftersterilization, each shell 180 was oriented horizontally and filled abouthalf full with nutrient agar medium 181, leaving a flat agar surface 182inside each shell extending the length of the shell. One end 183 of eachshell was sealed by dipping to a depth of 4 mm in paraffin 184, therebycausing the paraffin 184 to occupy about half the air space inside theshell. An embryo 185 was placed on the agar surface 182 (or "shelf")inside each shell, with the radicle 186 pointing toward the open end187, which was left exposed to the atmosphere. The resulting capsules188 were placed on their sides on a nutrient agar surface duringgermination.

Treatment (7): As in Treatment (6) except that, after placing an embryoon the "shelf" in each capsule, the open capsule ends were partiallysealed using PARAFILM in the same manner as described in Treatment (3).

All Treatments were incubated in covered 100 mm diameter Petri platesfor germination. Treatment (1) employed six plates containing sixembryos each. Treatments (2) through (7) employed three plates each, sixcapsules per plate. The plates were incubated for 35 days underconditions as described in Example 1. Data are tabulated in Table III.

                                      TABLE III                                   __________________________________________________________________________                            % Embryos                                                   % Normal                                                                            % Swollen                                                                           % Swollen                                                                           Completely                                                                           % Cotyledons                                   Treatment                                                                           Germinants                                                                          Hypocotyls                                                                          Cotyledons                                                                          Trapped                                                                              Trapped                                        __________________________________________________________________________    1 (Control)                                                                         72%   22%   6%     0%     0%                                            2     39%   44%   11%    6%    78%                                            3      6%   78%   45%   28%    61%                                            4     72%   17%   0%    17%    61%                                            5     12%   45%   0%    39%    61%                                            6     50%   34%   6%     6%    61%                                            7     34%   45%   6%    11%    79%                                            __________________________________________________________________________

Conclusions based on Table III and other observations were summarized asfollows:

(a) All Treatments lacking the PARAFILM-sealed end (Treatments (1), (2),(4), and (6)) exhibited a higher percent of normal germination,indicating a benefit of free exposure of the embryos to oxygen.

(b) Controls (Treatment (1)) as well as Treatment (4) exhibited thehighest percentages of normal germinants (72%), followed by Treatment(6) at 50% and Treatment (2) at 39%. Apparently, the combination oflight capsule weight and exposure of at least the radicle to oxygenduring germination was beneficial.

(c) No swollen cotyledons were seen in embryos that experiencedTreatment (4) or Treatment (5), indicating a benefit of lightweightcapsules.

(d) Treatments (3)-(6) exhibited the same percent of trapped cotyledons,even though the amount of medium in the capsules differed betweenTreatment (3), Treatments (4) and (5), and Treatment (6). Apparently,these capsule geometries are not optimal for allowing early releasetherefrom during gymnosperm germination.

(e) Partially sealing the radicle-end of the capsules with PARAFILMresulted in lower average lengths of hypocotyls and radicles (data notshown), probably demonstrating a slight negative effect of partial(although not excessive) physical obstruction of the radicle until itpenetrated the opening in the PARAFILM.

(f) Treatment (4) embryos experienced the same percent normal germinantsas the controls of Treatment (1). However, average lengths of hypocotylsand radicles, as well as average seedling weights (data not shown) ofTreatment (4) embryos, were less than with control embryos. Suchdecreased values, however, probably merely reflect the slightly greaterphysical restraints placed on a Treatment (4) embryo versus a "bare"embryo when undergoing germination.

EXAMPLE 5

In this Example, we evaluated enclosing the embryo in a porous tubeembedded in a nutrient-containing gel as an improved means forphysically securing an embryo inside a gel capsule without actuallyembedding an embryo directly in the gel. This method was investigatedbecause "shelf" capsules such as described in Treatment (4) of Example 4generally cannot be turned or handled without the embryo falling off thegel "shelf." The capsules tested in this Example also included a rigidexterior shell for additional physical protection. Securing the embryowas performed using a tube made of filter paper, where the filter paperserved as a liquid "bridge" between the gel and the embryo.

Individual sets of Norway Spruce embryos were subjected to one of thefollowing Treatments:

Treatment (1): "Control" as in Treatment (1) of Example 4.

Treatment (2): As in Treatment (4) of Example 4.

Treatment (3): Glass shells having 5.2 mm inside diameter were made asdescribed in Treatment (2) of Example 4. One end of each shell wassealed with an elastomeric septum, then the shells were sterilized. Eachshell was then filled with nutrient agar. Small paper tubes having 2.5mm inside diameter and about 5 mm long were made by cutting Whatman #1qualitative filter paper into 5 mm-wide strips, each of which was rolledaround a 2.5 mm outside diameter pin to form a paper tube. The tubeswere kept from uncurling by application of a small piece of label tape(2×8 mm). The tubes were autoclaved and sealed on one end by dipping inhot paraffin. Each tube was axially inserted sealed-end first in anindividual agar-containing glass shell until the open end of the tubewas flush with the opening of the shell. An embryo was inserted in eachpaper tube cotyledon-end first until the radicle tip was flush with thetube opening.

Treatment (4): Same as Treatment (3) except that the paper tubes were3.6 mm inside diameter instead of 2.5 mm inside diameter.

Each Treatment involved six sets having six embryos per set. InTreatments (2)-(4), the resulting capsules were placed on their sides onnutrient agar surfaces in sterile covered Petri plates and incubatedunder continuous light for 35 days at 23° C. Data are tabulated in TableIV.

                  TABLE IV                                                        ______________________________________                                                                         Trapped                                              % Normal    % Trapped    But Normal                                   Treatment                                                                             Germinants  Cotyledons (All)                                                                           Cotyledons                                   ______________________________________                                        1 (Control)                                                                           91%         --           --                                           3       20%         87%          19%                                          4       33%         75%          16%                                          ______________________________________                                    

After germination, observations and conclusions were summarized asfollows:

(a) The bare-embryo control (Treatment (1)) and the "shelf" capsule(Treatment (2)) produced nearly the same numbers of normal germinants;Treatments (3) and (4) involving embryos encased in paper tubes yieldedlower numbers of normal germinants. This may indicate that contact witha hydrated gel is more conducive to normal embryo development thancontact with paper. It is likely that using thinner paper tubes wouldyield higher numbers of normal germinants.

(b) In Treatments (2) to (4) involving encapsulation of the embryos, thecotyledons of a large percentage of germinants remained in the capsuleafter five weeks' incubation. This did not adversely affect normalcy inTreatment (2), but did in Treatments (3) and (4).

(c) Hypocotyl elongation was greatest in Treatments (1) and (2),followed by Treatment (4), then Treatment (3), indicating that the 2.5mm diameter paper tubes were too confining for the embryos. Radicleelongation was best in the controls (Treatment (1)), followed by the"shelf capsule" of Treatment (2).

(d) The 4 mm "shelf capsule" (Treatment (2)) appears to be an effectiveencapsulation method offering good embryo development, probably due toadequate exposure to oxygen.

EXAMPLE 6

In this Example, embryos were encapsulated in various gel formulationscomprising alginate and an emulsion of a perfluorocarbon to determinethe effects of such formulations on embryo germination and normaldevelopment.

A 30% emulsion of the perfluorocarbon FC-77(perfluorobutyltetrahydrofuran, 3M Co., St. Paul, Minn.) was prepared byadding to the FC-77 a sterile surfactant, Pluronic F-68 (1.5% w/vrelative to the FC-77), with the balance of the liquid being water.Pluronic F-68 is a polyoxypropylene polyoxyethylene polymer produced byBASF Corp., Parsippany, N.J. The mixture was emulsified using a Polytronhomogenizer (Brinkman Instruments Model #10 20 35D, generator #PT-DA3020/2TM) set to "High" for 30 seconds. Various amounts of the resultingemulsion were added to discrete concentrations of alginate in nutrientliquid. The purpose of using various concentrations of nutrient liquidwas to provide various degrees of compensation for the dilution causedby adding the liquid to the FC-77 emulsion. Mix ratios andconcentrations are as follows:

Treatment (1): Standard concentration of nutrient liquid containingalginate.

Treatment (2): A 1:1 v/v mixture of the 30% FC-77 emulsion with2×-concentrated nutrient liquid containing alginate.

Treatment (3): A 2:1 v/v mixture of the 30% FC-77 emulsion and3×-concentrated nutrient liquid containing alginate.

Treatment (4): A 3:1 v/v mixture of the 30% FC-77 emulsion and4×-concentrated nutrient liquid containing alginate.

Treatment (5): A 4:1 v/v mixture of the 30% FC-77 emulsion and5×-concentrated nutrient liquid containing alginate.

Treatment (6): "Control"; bare embryo placed on ix-concentrated nutrientliquid containing agar.

For Treatments (1)-(5), the mixtures of emulsion and nutrient liquidwith alginate were transferred immediately after preparation to asterile gas-washing bottle and oxygenated using sterile oxygen passingtherethrough for 30 minutes. The oxygenated mixtures were then placedindividually in a separatory funnel. Embryos were encapsulated in amanner similar to that disclosed in EPA '141 using 100 mM Ca(NO₃)₂ forcomplexing and nutrient liquid for rinsing. After encapsulation, thecapsules were placed on the surface of nutrient agar in covered Petriplates. For each Treatment, three plates were prepared, each containingsix embryos. All Treatments were incubated in continuous light at roomtemperature. A preliminary normalcy evaluation was made after two weeks'incubation and a final evaluation conducted after five weeks.

The data are tabulated in Table V and further illustrated in FIGS.9A-9F.

                  TABLE V                                                         ______________________________________                                                          2 Week %                                                            2 Week %  Growing   5 week %                                                                              5 Week %                                          Normal    Thru      Normal  Growing                                   Treatment                                                                             Germinants                                                                              Capsule   Germinants                                                                            Thru Capsule                              ______________________________________                                        1       6%        34%        0%     17%                                       2       0%        28%       12%     89%                                       3       0%        61%        0%     67%                                       4       12%       56%       28%     50%                                       5       6%        62%       23%     78%                                       6 (Control)                                                                           84%       --        88%     --                                        ______________________________________                                    

Results and conclusions are summarized as follows:

(a) As shown in Table V, the presence of oxygenated perfluorocarbons inthe form of an emulsion in an encapsulating hydrated gel aidsgermination and development of plant embryos from the alginate capsule,especially after five weeks. This is particularly evidenced by the factthat a large percentage of radicles were observed to elongate aftergermination in capsules containing perfluorocarbons, as shown in FIGS.9A and 9D.

(b) The percentage of swollen hypocotyls was approximately the same forTreatments (1)-(5) after two weeks' incubation, as shown in FIG. 9B.However, after five weeks' incubation, higher FC-77 emulsionconcentrations yielded fewer swollen hypocotyls, as shown in FIG. 9E.Since higher emulsion concentrations had correspondingly greateroxygen-absorbing ability, it appears that embryos encapsulated in gelshaving a higher emulsion concentrations developed more normally becausethey received more oxygen.

(c) The Controls (Treatment (6)) exhibited the best elongation ofhypocotyls. All encapsulated Treatments ((1)-(5)) exhibited almost equalelongation after both two weeks' and five weeks' incubation (FIG. 9C and9F). The fact that increasing the concentration of FC-77 had nosubstantial effect on hypocotyl length was not unexpected since previousstudies had shown that oxygen is not as limiting for hypocotylelongation as for radicle elongation. A better indicator of low oxygenin hypocotyls is swelling.

(d) After two weeks' incubation, the Control embryos (Treatment (6)) hadthe longest mean radicle length, as shown in FIG. 9C. Treatments (1)-(5)had somewhat variable radicle lengths. After five weeks, mean radiclelength in the Control was still the longest, but mean lengths inTreatments (1)-(5) were substantially equal to each other, as shown inFIG. 9F. The better growth of radicles after two weeks in Treatmentscontaining higher amounts of FC-77 correlates with the importance of theoxygen supply for radicle growth. The substantially equal growth ofradicles in Treatments (3)-(5) indicates that there is a concentrationof oxygen in a hydrated gel above which further improvement in radiclegrowth is not observed. However, as shown in the five week data of FIG.9F, radicle growth is not permanently inhibited at lower oxygen levels.Once the radicle grows out of an oxygen-limiting environment (i.e., thegel capsule), growth appears to accelerate.

(e) As shown in FIG. 9A, the percent of germinating radicles at twoweeks' incubation increased as the PFC concentration in theencapsulating gel was increased. After five weeks, the pattern changed,as shown in FIG. 9D. This indicates that more oxygen is preferred at theonset for germination when cells are beginning to rapidly divide andelongate.

(f) The data pertaining to embryos that grew through the capsules (TableV) illustrates that it would be preferable to physically restrain thecotyledons during germination. Such restraint keeps the burst capsule incontact for a time with the cotyledons rather than the hypocotyl (seeFIG. 4). The cotyledons, in turn, would carry the capsule upward out ofthe soil in a manner similar to the way a ruptured seed coat is carriedout of the soil. Then, when the cotyledons open, the capsule isdiscarded. The 4 mm-diameter shelf capsule tested as herein described inExample 5 is one example of a way to provide such restraint. As referredto herein, embryos that grew through the capsules are those that, asthey elongated, burst through both ends of the capsule, leaving thecapsule suspended around the hypocotyl (see FIG. 4). This condition canlead to swollen hypocotyls. However, there is no evidence that swollenhypocotyls decrease overall seedling survival.

(g) While there is no set pattern of normalcy in the encapsulatedTreatments tested in this Example, it appears that higher concentrationsof PFC yield more normal-appearing seedlings by supplying more oxygen tothe germinating embryo.

EXAMPLE 7

This Example was an evaluation of the ability of an alginate capsulecontaining an emulsion of PFC to support germination of embryos fromvarious species of conifers. The capsule material was prepared as twoseparate components that were combined to form the hydrated gel.

To prepare the alginate component, 333 mL of a 4.5% solution of ProtanalLF-60 alginate (Protan, Inc.) with conventional nutrients was prepared.The pH was adjusted to 5.7, and the solution was autoclaved for 20minutes.

The perfluorocarbon emulsion component was prepared as an emulsion of30% FC-77 and 1.5% Pluronic F-68, made as follows: approximately 200 mLof FC-77 and 70 mL of a 0.643% w/v Pluronic F-68 solution were eachautoclaved separately. After autoclaving, 30 mL of FC-77 was combinedwith the 70 mL of F-68 solution under sterile conditions and emulsifiedusing a Polytron homogenizer on the "High" setting for 30 seconds. To 80mL of the resulting emulsion were added 20 mL of the alginate component,yielding a final alginate concentration of 0.9%. The mixture was placedon a stir plate until a homogenous mixture was obtained. The resultinggel suspension was then transferred to a sterile gas-washing bottle andoxygenated under sterile conditions for 30 minutes.

To produce capsules around plant embryos, the oxygenated gel suspensionwas transferred to a sterile separatory funnel. The stopcock on theseparatory funnel was adjusted to form drops in a slow stepwise manner.Whenever a drop of the gel suspension formed at the tip of theseparatory funnel, a plant embryo was inserted into the drop usingsterile forceps, with the cotyledons pointing upward. The embryo wasfully immersed within the drop. The drop was then placed in a solutionof 100 mM Ca(NO₃)₂ with nutrients. This solution, termed a "complexingsolution," was adjusted to pH 5.7 and autoclaved prior to use. Thecapsules were allowed to harden in the calcium nitrate solution for 20minutes. Then, the calcium nitrate solution was discarded and thecapsules rinsed for five minutes with nutrient liquid before placementof the resulting capsules on the surface of nutrient agar in sterilecovered Petri plates.

Alginate solution lacking the PFC emulsion was prepared by combining oneliter of nutrient liquid with 15 g of Protanal LF-60 alginate. Afterautoclaving, the gel solution was oxygenated using a gas-washing bottleas described above (if required) and transferred to a sterile separatoryfunnel. Plant embryos were encapsulated in the alginate as describedabove.

Sixteen different combinations of embryo species and capsuleformulations were evaluated. The Treatments were as follows:

Treatment (1): "Control" wherein Norway Spruce bare embryos were placedon the surface of nutrient agar.

Treatment (2): Norway Spruce embryos encapsulated in non-oxygenatedalginate lacking PFC.

Treatment (3): Norway Spruce embryos encapsulated in oxygenated alginatelacking PFC.

Treatment (4): Norway Spruce embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (5): "Control" wherein Douglas Fir bare embryos were placed onthe surface of nutrient agar.

Treatment (6): Douglas Fir embryos encapsulated in non-oxygenatedalginate lacking PFC.

Treatment (7): Douglas Fir embryos encapsulated in oxygenated alginatelacking PFC.

Treatment (8): Douglas Fir embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (9): "Control" wherein Loblolly Pine bare embryos were placedon the surface of nutrient agar.

Treatment (10): Loblolly Pine embryos encapsulated in non-oxygenatedalginate lacking PFC.

Treatment (11): Loblolly Pine embryos encapsulated in oxygenatedalginate lacking PFC.

Treatment (12): Loblolly Pine embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (13): "Control" wherein Norway Spruce bare somatic embryoswere placed on the surface of nutrient agar.

Treatment (14): Norway Spruce somatic embryos encapsulated innon-oxygenated alginate lacking PFC.

Treatment (15): Norway Spruce somatic embryos encapsulated in oxygenatedalginate lacking PFC.

Treatment (16): Norway Spruce somatic embryos encapsulated in oxygenatedPFC-containing alginate.

All Treatments were incubated in continuous light at room temperaturefor five weeks, at which time they were-examined for germination andseedling development. The data are shown in Table VI and in FIGS. 10Aand 10B.

                                      TABLE VI                                    __________________________________________________________________________           % Normal                                                                            % That Grew                                                                           % Radicle                                                                            % Hypocotyl                                                                          % Germination                              Treatment                                                                            Germinants                                                                          Thru Capsule                                                                          Germination                                                                          Germination                                                                          Hyp. & Rad.                                __________________________________________________________________________    1 (Control)                                                                          92%   --      --     --     --                                         2       7%   28%     17%    92%    17%                                        3      17%   37%     45%    97%    45%                                        4      46%   87%     87%    100%   87%                                        5 (Control)                                                                          88%   --      --     --     --                                         6       3%   24%     21%    100%   21%                                        7       9%   24%     56%    94%    56%                                        8      30%   55%     59%    92%    59%                                        9 (Control)                                                                          92%   --      --     --     --                                         10      9%   37%     40%    95%    40%                                        11      3%   12%     15%    54%    15%                                        12     32%   70%     71%    98%    71%                                        13                                                                              (Control)                                                                          32%   --      --     --     --                                         14      3%   28%     30%    100%   30%                                        15     10%   34%     35%    100%   35%                                        16     21%   42%     47%    100%   47%                                        __________________________________________________________________________

The results can be summarized as follows:

(a) As shown in Table VI, the oxygenated PFC-containing alginate capsuleimproved germination and normalcy of all species tested, particularlyover germination and normalcy observed with capsules not containing anyPFC.

(b) For all species except Loblolly Pine, oxygenated alginate capsuleslacking PFC effected a higher number of normal germinants thannon-oxygenated capsules lacking PFC, as shown in Table VI.

(c) As shown in Table VI, the number of embryos that grew through bothends of the capsule was greater with oxygenated PFC-containing alginatecapsules than with the other types of capsules. This is an indicationthat the embryos germinating from oxygenated PFC-containing alginatecapsules had a high degree of vigor since the growing embryos werestrong enough to burst through both ends of the capsules. There is noevidence that this type of growth behavior is detrimental to the embryo.

(d) As shown in Table VI, hypocotyl germination was high in allTreatments (except with Loblolly Pine embryos) encapsulated inoxygenated alginate capsules lacking PFC. Radicle germination was bestwith oxygenated PFC-containing alginate encapsulated embryos for allspecies tested.

(e) As shown in FIG. 10A, swollen hypocotyls were still the mostprevalent abnormality, but swelling occurred less often with embryosencapsulated in oxygenated PFC-containing alginate.

(f) As shown in FIG. 10B, hypocotyl lengths increased as oxygenavailability in the capsule increased. This is indicated by the factthat the oxygenated PFC-containing alginate capsules yielded the longesthypocotyl lengths. Radicle lengths were greatest with embryosencapsulated in oxygenated PFC-containing alginate capsules, evensurpassing radicle lengths of bare embryos of Loblolly Pine.

EXAMPLE 8

In this Example, several candidate surfactants for use in making anemulsion of the perfluorocarbon were evaluated.

The methods used in this Example were substantially the same as used inExample 7 except that other surfactants and surfactant concentrationswere used. The study comprised six Treatments, as follows:

Treatment (1): PFC emulsion prepared using 1.5% Pluronic F-68 as asurfactant; Norway Spruce embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (2): PFC emulsion prepared using 4.0% egg albumin as asurfactant; Norway Spruce embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (3): PFC emulsion prepared using 1.5% sodium dodecyl sulfateas a surfactant; Norway Spruce embryos encapsulated in oxygenatedPFC-containing alginate.

Treatment (4): Norway Spruce embryos encapsulated in oxygenated alginatelacking the PFC emulsion.

Treatment (5): Norway Spruce embryos encapsulated in non-oxygenatedalginate lacking the PFC emulsion.

Treatment (6): "Control" wherein Norway Spruce bare embryos were placedon the surface of nutrient agar.

All Treatments utilized Norway Spruce zygotic embryos and each consistedof six encapsulated embryos prepared per covered Petri plate, six platesfor each Treatment. All plates were incubated in continuous light atroom temperature for five weeks, at which time the germinants wereevaluated for germination success and other parameters. The results areshown in Table VII and in FIGS. 11A and 11B.

                                      TABLE VII                                   __________________________________________________________________________          % Normal                                                                            % Growth                                                                             % Radicle                                                                            % Hypocotyl                                                                          % Germination                                Treatment                                                                           Germinants                                                                          Thru Capsule                                                                         Germination                                                                          Germination                                                                          Hyp. & Rad.                                  __________________________________________________________________________    1     56%   94%    94%    100%   94%                                          2     70%   86%    86%     97%   86%                                          3      0%    0%     0%     0%     0%                                          4     15%   57%    59%    100%   59%                                          5     24%   35%    41%     89%   35%                                          6 (Control)                                                                         100%  --     --     --     --                                           __________________________________________________________________________

Conclusions drawn from the results can be summarized as follows:

(a) As shown in Table VII, Treatment (3), sodium dodecyl sulfate is notan effective surfactant in that it caused mortality of all embryos incontact with it.

(b) As shown in Table VII, Treatments (2) and (1), respectively, eggalbumin and Pluronic F-68 are both effective surfactants for PFCs suchas FC-77. Egg albumin produced more normal germinants, but Pluronic F-68yielded more germinated embryos.

(c) As shown in Table VII and FIG. 11A, oxygenated PFC-containingalginate capsules yielded a higher level of normalcy and a higher totalnumber of germinants than seen with alginate capsules lacking PFC,whether oxygenated or not.

(d) As expected, bare embryos grown on agar produced the most normalgerminants.

(e) Treatment (1) yielded the most embryos that grew through the capsule(Table VII).

EXAMPLE 9

In this Example, the ability of various perfluorocarbons to supplyoxygen to encapsulated embryos was evaluated.

The methods employed in this Example are the same as those in Example 7except that several different perfluorocarbons were used. The variousTreatments tested were as follows:

Treatment (1): Norway Spruce embryos encapsulated in oxygenated alginatecontaining an emulsion of 30% FC-77 plus 1.5% Pluronic F-68.

Treatment (2): Norway Spruce embryos encapsulated in oxygenated alginatecontaining an emulsion of 30% perfluorodecalin (another type of PFC) and1.5% Pluronic F-68.

Treatment (3): Norway Spruce embryos encapsulated in oxygenated alginatecontaining an emulsion of 30% perfluorotributylamine (another type ofPFC) and 1.5% Pluronic F-68.

Treatment (4): Norway Spruce embryos encapsulated in oxygenated alginatelacking PFC.

Treatment (5): Norway Spruce embryos encapsulated in non-oxygenatedalginate lacking PFC.

Treatment (6): "Control" wherein Norway Spruce bare embryos were placedon the surface of nutrient agar.

All Treatments utilized Norway Spruce zygotic embryos and each consistedof six covered Petri plates containing six encapsulated embryos perplate. Treatments were incubated in continuous light at room temperaturefor five weeks, after which germination success and other parameterswere evaluated. Results are tabulated in Table VIII and shown in FIGS.12A and 12B.

                                      TABLE VIII                                  __________________________________________________________________________          % Normal                                                                            % Growth                                                                             % Radicle                                                                            % Hypocotyl                                                                          % Germination                                Treatment                                                                           Germinants                                                                          Thru Capsule                                                                         Germination                                                                          Germination                                                                          Hyp. & Rad.                                  __________________________________________________________________________    1     69%   92%    95%    100%   97%                                          2     35%   70%    77%    100%   77%                                          3     61%   81%    86%    100%   86%                                          4     34%   56%    56%    100%   56%                                          5     29%   60%    65%    100%   65%                                          6 (Control)                                                                         97%   --     --     --     --                                           __________________________________________________________________________

The conclusions can be summarized as follows:

(a) As shown in Table VIII, it appears that perfluorodecalin (Treatment(2)) does not produce as many normal germinants as does FC-77 (Treatment(1)) and perfluorotributylamine (Treatment (3)). However,perfluorodecalin produces substantially the same number of normalgerminants as oxygenated alginate lacking PFC (Treatment (4)). Thiscould be due to a short half life of the perfluorodecalin emulsion.

(b) It appears that FC-77 is the preferred perfluorocarbon among thosetested in this Example for use in analogs of botanical seed, at least ofconifers.

(c) As expected, bare embryos (Treatment (6)) had the highest percentageof normal germinants, as shown in Table VIII. Treatment (5), involving anon-oxygenated alginate capsule, had the lowest percent of normalgerminants.

(d) Treatment (1) had the highest percent of embryos growing through thecapsule (Table VIII).

(e) All hypocotyls in all Treatments germinated (Table VIII). Thepercentages of radicle germination and germination of both radicle andhypocotyl were highest in Treatments having perfluorocarbon emulsions inthe alginate, as shown in Table VIII.

(f) As shown in FIG. 12A, Treatments (2), (4), and (5) yieldedapproximately two times more abnormalities than the other threeTreatments, where swollen hypocotyls were the most prevalentabnormality.

(g) Of the encapsulated embryos, radicle lengths and hypocotyl lengthswere longest when embryos germinated from oxygenated PFC-containing gelcapsules (FIG. 12B).

EXAMPLE 10

The objective in this Example was two-fold: (1) to evaluate the effecton normal germination of an alginate capsule containing only surfactantand no PFC; and (2) to evaluate the effect on normal germination ofencapsulating embryos in non-oxygenated PFC-containing alginatecapsules.

The methods used for encapsulating embryos are as described above inExample 7. Individual sets of Norway Spruce embryos were subjected toone of the following Treatments:

Treatment (1): Embryos encapsulated in oxygenated alginate containingFC-77 emulsion, according to Example 7.

Treatment (2): Embryos encapsulated in nonoxygenated alginate containingFC-77 emulsion.

Treatment (3): Embryos encapsulated in oxygenated alginate lacking PFCbut containing 1.5% Pluronic F-68.

Treatment (4): Embryos encapsulated in non-oxygenated alginate lackingPFC but containing 1.5% Pluronic F-68.

Treatment (5): Embryos encapsulated in non-oxygenated alginate lackingboth PFC and surfactant.

Treatment (6): Embryos encapsulated in oxygenated alginate lacking bothPFC and surfactant.

Treatment (7): "Control" wherein bare embryos were grown on the surfaceof nutrient agar.

The concentration of Pluronic F-68 in the alginate capsules used inTreatments (3) and (4) was the same as used in Treatments (1) and (2).Each Treatment comprised six covered Petri dishes, each containing sixembryos. All Treatments were incubated in continuous light at roomtemperature for 35 days. Results are shown in Table IX and FIGS. 13A and13B.

                                      TABLE IX                                    __________________________________________________________________________          % Normal                                                                            % Growth                                                                             % Radicle                                                                            % Hypocotyl                                                                          % Germination                                Treatment                                                                           Germinants                                                                          Thru Capsule                                                                         Germination                                                                          Germination                                                                          Hyp. & Rad.                                  __________________________________________________________________________    1     35%   82%    82%    100%   82%                                          2     27%   52%    49%    100%   49%                                          3     18%   36%    36%     97%   36%                                          4      6%   19%    20%     95%   20%                                          5      9%   28%    28%    100%   28%                                          6      6%   34%    34%    100%   34%                                          7 (Control)                                                                         94%   --     --     --     --                                           __________________________________________________________________________

The results and conclusions can be summarized as follows:

(a) As shown in Table IX, both oxygenated and non-oxygenatedPFC-containing alginate capsules (Treatments (1) and (2)) yielded moregerminants and a higher percent of normal germinant than non-PFCcontaining alginate capsules.

(b) As shown in Table IX, Pluronic F-68, in an alginate capsule lackingPFC, appears to increase germination when the capsule has beenoxygenated (Treatment (3)), and to decrease germination when the capsuleis non-oxygenated (Treatment (4)).

(c) Of the capsule formulations tested, the oxygenated alginate capsulecontaining PFC emulsion appears to be the best.

(d) It appears that the benefit of adding an emulsion of PFC to thealginate capsule is derived from the presence of the PFC and not merelythe surfactant therein.

(e) Treatment (1) exhibited the highest percent of embryos that grewthrough the capsule (Table IX).

(f) Hypocotyl germination was high with all Treatments (Table IX).Treatment (1) exhibited the highest values of percent germination ofboth radicle and hypocotyl.

(g) The only types of malformations observed were swollen hypocotyls andtwisted cotyledons (FIG. 13A).

(h) The controls (Treatment (7)) exhibited the longest radicles andhypocotyls (FIG. 13B). Treatment (1) embryos exhibited the longesthypocotyl lengths of the encapsulated embryos, as well as the longestradicle lengths.

Having illustrated and described the principles of the invention inmultiple embodiments and examples, it should be apparent to thoseskilled in the art that the invention can be modified in arrangement anddetail without departing from such principles. We claim allmodifications coming within the spirit and scope of the followingclaims.

We claim:
 1. A method for manufacturing an analog of a botanic seedcomprising:preparing a liquid hydrated gel comprising a perfluorocarboncompound so as to enable the gel to acquire a concentration of molecularoxygen that is higher than a concentration of molecular oxygen thatwould otherwise be present in an otherwise similar gel, that lacks theperfluorocarbon compound, solely by absorption of oxygen from theatmosphere at standard temperature and pressure; encapsulating a unit oftotipotent plant tissue in the gel; and curing the gel.
 2. A method formanufacturing an analog of a botanic seed as recited in claim 1including the step of incorporating molecular oxygen in the gel bypassing oxygen gas through the gel before curing the gel.
 3. A methodfor manufacturing an analog of a botanic seed as recited in claim 1including the step of incorporating molecular oxygen in the gel bypassing oxygen gas through the gel after curing the gel.
 4. A method formanufacturing an analog of a botanic seed as recited in claim 1 whereinthe step of preparing the gel comprises incorporating theperfluorocarbon compound in the gel.
 5. A method for manufacturing ananalog of a botanic seed as recited in claim 4 wherein the step ofpreparing the gel comprises emulsifying the perfluorocarbon compound inthe presence of a surfactant to form a suitably stabilized emulsion ofthe perfluorocarbon compound, and adding the emulsion to the gel.
 6. Amethod for manufacturing an analog of a botanic seed as recited in claim1 including the step of enclosing the gel with a rigid shell.